Organic compounds -- part of the class 532-570 series – Organic compounds – Amino nitrogen containing
Reexamination Certificate
1999-09-03
2001-01-09
Henley, III, Raymond (Department: 1614)
Organic compounds -- part of the class 532-570 series
Organic compounds
Amino nitrogen containing
Reexamination Certificate
active
06172261
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention in the field of chemistry and biochemistry relates to the synthesis and use of novel polyamine transport (PAT) inhibitor compounds with pharmacological or agricultural uses and as probes for biochemical assays or for purification of selected polyamine binding targets. As drugs, these compounds are used to treat disorders of undesired cell proliferation, primarily cancer, alone or combined with other agents such as polyamine synthesis inhibitors. An assay employing some of these compounds is useful for monitoring polyamine uptake or transport (PAT) and allows analysis of binding sites for polyamines or for other basic ligands on a variety of molecules. This invention also relates to the synthesis and use of novel polyamine combinatorial libraries. These libraries are used to discover compositions that inhibit PAT and/or that bind to a cellular polyamine transporter (PATr). Various members of these libraries or compounds discovered through use of the libraries have utility as drugs, agricultural chemicals, and as probes. This invention also identifies key elements that comprise the polyamine binding sites of membrane as well as soluble proteins.
2. Description of the Background Art
Polyamines are ubiquitous molecules that provide a “buffer” system for the cell by modulating the activities of proteins, RNA, DNA, and lipids. Polyamines may play a direct role in apoptosis. Mammals and other organisms have an active polyamine uptake and recycling system that complements their polyamine synthetic capabilities. Because polyamines modulate such a large range of molecules and cellular activities, polyamine analogues, as disclosed herein, offer novel approaches for targeting a variety of disease states, particularly cancer, and also provide unique tools to monitor cellular activities.
Polyamines and Cancer
The potential of polyamines as anticancer agents has been long recognized. Polyamines affect chromatin structure in eukaryotes and prokaryotes by binding specifically to DNA (Balasundaram, D. et al.,
Mol. Cell. Biol.
100:129-140, 1991) so that condensation occurs when the binding sites on DNA are saturated. Acetylation of polyamines and histones lowers their affinity for DNA and is believed to occur in tandem to alter the structure and function of the nucleosome, thus regulating DNA replication and transcription by loosening DNA at the ends of the core particle. Because polyamines are absolutely essential for DNA replication, they are of interest in the treatment of cancer. Particular interest has been focused on preventing cell proliferation by lowering intracellular polyamine levels. Polyamine analogues as described herein are useful for preventing or treating cancer and other proliferative diseases by acting at a number of different levels. The present invention focuses on the inhibition of PAT. Other targets include induction of spermine/spermidine acetyltransferase (SSAT), hypusine modification, and other proteins that inhibit the cell cycle or induce apoptosis.
The Polyamine Transporter (PATr)
The increased demand for polyamines by rapidly growing, transformed cancer cells is only partially met by an increased rate of synthesis. To exploit this increased need for polyamines, synthesis inhibitors have been sought. Additionally, lowering polyamine concentrations can result in aberrations in chromatin structure leading to cell death or inhibition of proliferation (Quemener, V. et al.,
Anticancer Res.
14:443-448, 1994; Porter, C. W. et al.,
Cancer Res.
53:581-586, 1993). It has become increasingly apparent that the initial disappointing results observed in the clinic with polyamine synthesis inhibitors arises from compensatory increases in transport of polyamines by a specific active transport system (Seiler, N. et al.,
Int. J. Biochem.
22:211-218, 1990; Seiler, N. et al.,
J. Biochem. Cell. Biol.
28:843-861, 1996). The promising results observed in cell culture with a suicide substrate inhibitor of ornithine decarboxylase, &agr;-difluoromethylornithine (DFMO), or with an inhibitor of S-adenosylmethionine decarboxylase, methylglyoxal bis(guanylhydrazone) (MGBG) did not transfer to human clinical trials (Schecter, P. J. et al.,
In Inhibition of Polyamine Metabolism. Biological Significance and Basis for New Therapies;
McCann, P. P. et al., eds; 1987, pp 345-364). Since the only two avenues for carbon transfer into polyamine pools are synthesis or transport, simultaneous inhibition of both of these pathways is considered by the present inventors to be a promising anti-cancer therapeutic approach.
A study confirming the validity of this chemotherapeutic approach used transplanted murine L1210 leukemia cells that were deficient in PAT. Mice transplanted with the wild-type L1210 cancer cells (with intact PAT) died after 12 days, even when treated with DFMO. In contrast, DFMO mice transplanted with PAT-deficient L1210 cells lived longer than 60 days (Ask, A. et al.,
Cancer Lett.
66:29-34, 1992). These authors also showed that treatment of mice harboring wild-type L1210 cells with a combination of (1) DFMO (2) a low polyamine diet and (3) antibiotics (which decrease polyamine production by gut flora) resulted in prolonged survival compared to treatment with DFMO alone.
Augmented PAT into cancer cells promotes cell killing. J. L. Holley et al. (
Cancer Res.
52:4190-4195, 1992) showed up to a 225-fold increase in cytotoxicity of a chlorambucil-spermidine conjugate compared to chlorambucil alone. A series of nitroimidazole-polyamine conjugates were also effective (Holley, J. L. et al.,
Biochem. Pharmacol.
43:763-769, 1992). Others showed that mice infected with a multi-drug resistant strain of malaria were cured by treatment with a chloroquinoline-putrescine conjugate (Singh, S. et al.,
J. Biol. Chem.
272:13506-13511, 1997). Thus, the effectiveness of cytotoxic compounds could be enhanced by their conjugation with polyamines. These effects may have been due to the exploitation of the PAT system to deliver these compounds into cancer cells. The present invention is therefore directed in part to rapid and efficient testing of many different conjugates between polyamines and known drugs for their transport into cells. Furthermore, as described below, this invention combines the cytotoxic properties of known drugs with the facilitated transport of polyamines, which relies on the present inventors' discoveries surrounding the PATr described herein. By accessing the database of structure-activity-relationships (SARs) of PATr substrates, the present inventors are able to predict the transportability of a novel chemical entity or a novel polyamine conjugate.
Polyamine Transport (PAT) Assays
There is no known high-throughput assay for measuring PAT. A radiochemical assay is used for biochemical analysis of transport and has been used to study PAT in yeast and a variety of mammalian cells (Kakinuma, Y. et al.,
Biochem. Biophys. Res. Comm.
216:985-992, 1995; Seiler, N. et al.,
Int. J. Biochem. Cell Biol.
28:843-861, 1996). See, for example Huber, M. et al.
Cancer Res.
55:934-943, 1995.
The radiometric assay uses radiolabeled polyamines such as putrescine, spermidine or spermine, but, due to the low signal, large numbers of adherent or non-adherent cells are required. Additional care is required with spermine due to its non-specific adsorption to cells and plastics. Cells are mixed with the test compounds and the radiolabeled polyamine to initiate the assay. The cells are incubated for 1-60 minutes, depending on cell type. The assay is terminated by removal of the medium and cooling the plates to 4° C. The cells are then washed with cold medium three times, dissolved in 0.1% sodium dodecyl sulfate and the radioactivity in solution is then determined by scintillation counting. This assay is difficult to scale up to a high throughput procedure due to the low signal from the radiolabel and the handling requirements inherent in procedures with radioactivity.
Combinatorial Approaches to Polyamines and Analogues
Co
Bergstrom Donald E.
Burns Mark R.
O'Day Christine L.
Vermeulin Nicolaas M. J.
Webb Heather K.
Henley III Raymond
Morrison & Foerster / LLP
Oridigm Corporation
LandOfFree
Polyamine analogues as therapeutic and diagnostic agents does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Polyamine analogues as therapeutic and diagnostic agents, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Polyamine analogues as therapeutic and diagnostic agents will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2480944